2'-Azido-2'-deoxyuridine- 5'-diphosphate (N3UDP) has previously been shown to be a stoichiometric mechanism based inhibitor of RNR. Inactivation of RNR is accompanied by loss of the tyrosyl radical on the R2 subunit concomitant with formation of a new nitrogen centered radical. The Xband EPR spectrum of this stable radical species exhibits a triplet hyperfine interaction of -25 G arising from one of the three nitrogens of the azide moiety of N3UDP and a doublet hyperfine interaction of 6.3 Gauss which has been proposed to arise from a proton. High frequency (139.5 GHz) EPR spectroscopic studies of this nitrogen centered radical have resolved the three principal g-values: g I I = 2.01557, 922 2.00625, and 933 = 2.00209. In addition, the nitrogen hyperfine splitting along 933 is resolved (A33= 3 1.0 G) and upper limits (- 5 G) can be placed on both A I , and A 22 . Comparison of these g and A [unreadable]values with those of model systems in the literature suggests a structure for the radical, X-N--S-CH2-, in which SCH2 is part of a cysteine residue of RI and X is either a non-protonated sulfur, oxygen or carbon moiety. Use of an E. coli strain that is auxotrophic for cysteine and contains the nucleotide reductase gene allowed [p-2H]-cysteine labeled RNR to be prepared. Incubation of this isotopically labeled protein with N3UDP produced the radical signal without the hyperfine splitting of 6.3 G, indicating that this interaction is associated with a proton from the -S-CH2-component of the proposed structure. These results establish that the nitrogen centered radical is covalently attached to a cysteine, probably C225, of the RI subunit of RNR. Site-directed mutagenesis studies with a variety of RI mutants in which each cysteine (439, 462, 754 & 759) was converted to a serine reveal that X cannot be a substituted sulfur. A structure for the nitrogen centered radical is proposed in which X is derived from 3'-keto-2'-deoxyuridine 5'-diphosphate, an intermediate in the inactivation of RNR by N3UDP. Specifically, X is proposed to be the Y-hydroxyl oxygen of the deoxyribose moiety.